期刊
NATURE
卷 541, 期 7636, 页码 242-+出版社
NATURE RESEARCH
DOI: 10.1038/nature20599
关键词
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资金
- LCLS Ultrafast Science Instruments (LUSI) project - US Department of Energy, Office of Basic Energy Sciences
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-76SF00515]
- NSF-STC BioXFEL [NSF-1231306]
- NIH Intramural Research Program of NCI
- NIH Intramural Research Program of CIT
- NIH Intramural Research Program of NHLBI
- NIH Intramural Research Program of US Department of Energy, Office of Biological and Environmental Research [DE-AC02-06CH11357]
- European Research Council [ERC-2013-SyG 609920]
- BMBF [05K16GU1]
Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform(1-3). A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time(4). Here we use femtosecond X-ray free electron laser (XFEL) pulses(5,6) to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro-and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.
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